Fabrication and compatibility evaluation of polycaprolactone/hydroxyapatite/collagen-based fiber scaffold for anterior cruciate ligament injury

Knee injuries are musculoskeletal system injuries, including the Anterior Cruciate Ligament (ACL). ACL injuries are most common in athletes. This ACL injury necessitates biomaterial replacement. It is sometimes taken from the patient's tendon and a biomaterial scaffold is used. The use of biomaterial scaffolds as artificial ACLs remains to be investigated. The purpose of this study is to determine the properties of an ACL scaffold made of polycaprolactone (PCL)–hydroxyapatite (HA) and collagen with various composition variations of (50 : 45 : 5), (50 : 40 : 10), (50 : 35 : 15), (50 : 30 : 20), and (50 : 25 : 25) wt%. The scaffold was created using the electrospinning method with a voltage of 23 kV, a needle–collector distance of 15 cm, and a solution flow rate of 2 mL h−1. The average fiber diameter in all samples was less than 1000 nm. The model with the best characterization was PCL : HA : collagen with a weight-to-weight (wt%) ratio of 50 : 45 : 5 and an average fiber diameter of 488 ± 271 nm. The UTS and modulus of elasticity for braided samples were 2.796 MPa and 3.224 MPa, respectively, while the non-braided samples were 2.864 MPa and 12.942 MPa. The estimated time of degradation was 9.44 months. It was also revealed to be non-toxic, with an 87.95% viable cell percentage.


Introduction
Knee injuries are musculoskeletal system injuries that frequently occur alongside back injuries. This injury affects 48 per 1000 patients per year in Europe. In this instance, 9% of the ligaments, including the Anterior Cruciate Ligament, were damaged (ACL). Most ACLs rupture during sports activities. The incidence is highest between the ages of 15 and 25 in athletes who rotate the knee joint, such as those who play soccer, basketball, European handball, and volleyball. The injury is caused by valgus or external rotational trauma with a slightly bent knee. 1 The severity of an ACL tear determines the treatment for the injury. If the injury can impair quality of life, especially in athletes who are actively moving, the treatment is ACL surgery. A torn ACL is typically treated with a gra harvested from the patient (autogra). However, autogra therapy poses a risk of tissue damage and tends to lengthen surgical procedures. Consequently, tissue engineering is utilized to develop an efficient method for ACL reconstruction.
In the last four decades, tissue engineering has emerged as an area of study. Tissue engineering aims to restore, maintain, or improve the function of damaged or lost tissues as a result of physiological, pathological, or mechanical conditions or trauma by developing biological replacements or reconstructing the tissues. 2 Combining biomaterials such as scaffolds, stem cells, and growth factors will produce medically applicable tissue engineering products. The three factors, known as the tissue engineering triad, are inseparable: scaffold, stem cells, and growth factors. They resemble the regeneration of cells, tissues, and organs that occurs naturally.
Through the development of scaffolds, materials science has played a signicant role in this process. The scaffold is a medium or framework that provides an environment for stem cells or other cells to adhere, proliferate, and differentiate, ultimately resulting in the formation of the desired tissue. The scaffold must be designed with the appropriate properties for its intended function, and its surface must have the correct morphology for cell attachment and differentiation. 3 Engineered tissue for ACL injuries must possess the same biomechanical properties as the original ACL tissue in order to reconstruct the injury properly. Biopolymer is an excellent material that is frequently used to reconstruct damaged tissue. Biopolymers typically used for tissue reconstruction must also possess excellent biodegradability. 4 However, biodegradable biopolymers must be carefully considered in terms of biocompatibility, as these properties can have toxic effects during degradation. 5 One of the biopolymers employed in tissue engineering is polycaprolactone (PCL). With a modulus of elasticity between 0.21 and 0.44 GPa, 6 PCL is very ductile and provides low stiffness. PCL is a polymer with excellent biocompatibility and degradation characteristics. PCL has a signicantly lower rate of degradation than PLA, PGA, and PLGA. 7 Two years are required for PCL to completely degrade. 8 PCL has additional benets, such as reducing local acidication and inammation. 9 Vascular, bone, cartilage, nerve, skin, and esophageal tissue are among the many applications of PCL in tissue engineering. 10 To give PCL bioactive properties, hydroxyapatite must be added. Hydroxyapatite (HA) is the most abundant mineral in human bone. HA is frequently employed in biomedical implant applications or for tissue reconstruction and regeneration. HA possesses excellent bioactivity and osteoconductive properties. It is anticipated that the addition of HA to ACL reconstruction will stimulate cell growth in the femur and tibia, which are the scaffold's attachment sites so that the bone can integrate with the scaffold. HA is the most thermodynamically stable calcium phosphate ceramic compound in solution; its pH, temperature, and chemical composition are most similar to those of physiological uids. 11 In addition to HA, collagen must be added in order to match the Extracellular Matrix (ECM). Collagen has been used extensively to promote cell growth and differentiation during tissue formation. As the most abundant protein in the human body, collagen serves as physical support in tissues by occupying intercellular spaces, not only as structural support for regulating cells in connective tissue but also as a mobile, dynamic, and exible substance that is essential for cellular behavior and network function. 3 Collagen is also bio-inductive, possesses mechanical properties that are compatible with ECM, and is biodegradable, which makes it a popular choice for clinical applications. Multiple studies have demonstrated that collagen can enhance cell adhesion, promote bone cell proliferation, and enhance osteogenic cell differentiation. In addition, collagen dramatically increases the initial adhesion of the periosteal segment, which facilitates cell development and handling efficiency during implantation.
On the basis of the preceding information, the purpose of this study was to investigate the effect of variations in the composition of HA and collagen on the PCL-HA-collagen scaffold on a number of characteristics, including ber surface morphology, ber size, mechanical strength, degradation rate, and cytotoxicity. Electrospinning is used to create bers because the ACL is anatomically composed of dense bands of collagen bers. Electrospinning produces bers with advantageous characteristics, such as high porosity, a large surface area, and continuous and quite long lengths. 12

Materials and methods
This study divided the production of ber samples into three stages. The initial step involved the preparation of PCL-HAcollagen solutions of varying compositions. Stage 2 was the electrospinning process with constant process parameters for all samples, including a voltage of 23 kV, a needle-to-collector distance of 15 cm, and a ow rate of 2 mL h −1 . The third stage was sample characterization, which included psychochemical characterization with FTIR spectrometer and Scanning Electron Microscope (SEM), mechanical properties, degradation rate, and cell viability evaluation using MTT assay. Fig. 1 provides a schematic representation of the study's work process.

Preparation of PCL
2.2.2. Manufacturing of ber through an electrospinning process. A 10 mL syringe was lled with the stirrer solution. The syringe was then connected to the electrospinning instrument. A at-shaped collector was used in this electrospinning process. The bers were collected using a at collector covered in aluminum foil. The power supply's connecting cable was connected to the syringe needle. To ensure proper voltage transmission, the connecting wire must be properly attached to the needle.
In addition, ber formation by electrospinning was carried out. The electrospinning process used a high voltage of 23 kV, a distance of 15 cm between the tip of the needle and the collector, and a ow rate of 2 mL h −1 . The solution began to ow through the bers that produce needles. The electric eld inuences the bers' trajectory, causing them to deposit on the aluminum foil. The procedure was repeated until the syringe was empty. Electrospun bers were the end result of the electrospinning process. They were cut to various sizes based on the requirements of the test.

Characterization
2.3.1. Characterization of functional groups of PCL-HAcollagen scaffold using Fourier transform infra-red (FTIR) spectrophotometer. The electrospun ber samples were then tested for functional groups using the Shimadzu IRTracer-100 FTIR at a wavenumber of 400-4000 cm −1 . The FTIR test results show the relationship between % transmission and wavenumber spectrum (cm −1 ). The absorbance band formed in the sample's infrared spectrum was compared to standard data and the spectrum of the comparison compound was used for functional group analysis.
2.3.2. Surface morphology and diameter of PCL-HAcollagen scaffold ber using scanning electron microscope (SEM). Surface morphology test of ber samples using SEM Hitachi FLEXSEM 1000. The sample was gold-coated before measurements (Au). The ber diameter was determined by analyzing SEM observation images with the ImageJ application. Calibration of the image pixel with the reference size should be the rst step. The reference size is typically shown on the SEM image alongside a scale indicating the magnication level. The diameter of the average ber can be measured using ImageJ soware supported by Originlab to generate a sample diameter distribution plot. The light-dark area fraction is another parameter that can be analyzed microscopically from the surface structure. The dark fraction represents space, while the light area represents the formed ber. To begin, the SEM image is segmented using a threshold to improve the denition of nanober and background. Aer adjusting the threshold, the area fraction analysis in question can be performed using Image-Histogram J's feature. The histogram shows a value of 0 for dark areas and a value of 255 for light areas.
2.3.3. Measurement of mechanical properties of PCL-HAcollagen scaffold ber. The mechanical test was performed to determine the sample's mechanical strength, specically the modulus of elasticity, tensile strength, and elongation. Fiber samples were cut into dogbone shapes measuring 2 cm × 6 cm, and some were braided. Here we compare the design of scaffold using dogbone shapes (unbraided) and braided scaffold to understand in more detail the mechanical properties of the fabricated samples. It is predicted that the sample with dogbone and braided scaffold should have different in mechanical properties due to difference in how the scaffold receive a mechanical stress. 13 A micrometer was used to measure the sample's thickness. The sample is then attached to the Shimadzu AGS 1kNX universal testing machine and a loading force is applied. The sample is then pulled until it breaks. Stress variations were measured during the tensile process. The slope of the linear region of the stress-strain curve at the maximum loadto-failure point was used to calculate tensile strength (N mm −1 ). Tensile strength characterization was performed three times on all unbraided and braided samples. The Ultimate Tensile Strength (UTS) value is calculated using eqn (1) and Young's modulus or modulus of elasticity (E) value is calculated using eqn (2), or it is calculated from the gradient value of the stress-strain linear curve and the elongation value using eqn (3). Before being immersed in PBS (W 0 ) solution, samples of ber were weighed to determine their weight. The samples were submerged in a pH 7.4 PBS solution and incubated at 37°C for 7, 14, 21, and 28 days. Following immersion, samples were weighed on days 7, 14, 21, and 28 to determine their nal weight (W t ). Each measurement was carried out thrice. Using eqn (4), the percentage of weight loss was determined. In addition, the degradation rate and estimates of the completely degraded scaffold in the solution are calculable.

Cytotoxicity test for PCL-HA-collagen scaffold.
A cytotoxicity test is a test performed to determine the levels of toxicity in prepared samples. This evaluation employs broblast cell culture (cell line BHK 21). The cells were incubated on Eagle's medium, or until a single conuent layer formed on the respective wall. Thus, the cells seeded onto scaffold for 24 hours. The remaining serum was then cleaned with dimethyl sulfoxide (DMSO) aer the removal of the medium. To prevent colonization, the cells were then separated using 0.25 percent vergence trypsin.
In addition, Eagle media cells and MTT reagent were added, then transferred to a microplate 96 and incubated at 37°C for four hours. Each measurement was performed thrice. To stop the reaction, DMSO was added to each well, which was then vortexed for 5 minutes to achieve homogeneity. Repeating each sample four times. With the aid of an ELISA reader, the optical density (OD) was determined. Eqn (5) was used to calculate cell viability.
ð% cell viabilityÞ ¼ OD treatment À OD media OD cell À OD media Â 100% (5) 3. Results The bers from the aluminum foil were removed. It was then sized appropriately for the various tests. Fibers were separated for mechanical property tests (Fig. 2b). Fig. 2c demonstrates the braided bers.

Analysis of the PCL-HA-collagen scaffold functional groups from the Fourier transform infra-red (FTIR) spectrum
The interaction of infrared radiation and molecular vibrations produces the Fourier Transform Infra-Red (FTIR) spectrum.
Each type of molecular vibration has a unique value expressed by a wavenumber (cm −1 ). Fig. 3 depicts the PCL-HA-collagen scaffold spectrum. group at wavenumber 960.55 cm −1 . 14 Additional markers include the CO 3 2− group at wave numbers 1415.75-1417.68 and 1463 cm −1 . 14 In addition, the collagen marker is identied by an amide III group (N-H bend) in the wave number region of 1240.23 cm −1 .  The ber surface morphology was examined using a 1000× magnication Scanning Electron Microscope (SEM) (Fig. 4).
Because the collector used is not drum-shaped, the bers tend to pile up and arrange themselves randomly in all samples. Furthermore, it can be caused by the solution being exposed to high voltages that are not optimally attracted, causing instability and random orientations. The shape of the aligned ber is more advantageous than that of the random ber. Fibers with aligned shapes promote cell proliferation and improve sample mechanical properties. 15 Furthermore, all samples had rough surface morphology. This could be because hydroxyapatite and collagen were added. According to Han's 2015 research, the morphology of PCL samples with hydroxyapatite and collagen was coarser than PCL without hydroxyapatite and collagen. 16 The formation of beads in Fig. 4 can be attributed to the solution's low dielectric properties. DMF solvent can increase the dielectric properties, preventing the formation of beads. Based on Fig. 4, the diameter of the bres is presented in Table 2. Table 2 revealed that the ber diameter varied, with the diameter decreasing as the collagen content increased. The light-dark area fraction was also determined using SEM analysis that we have tabulated in Table 3.
The value of the dark fraction represented an area completely void of ber. This value was affected by the collagen content percentage of the scaffold. Collagen is essential for hydroxyapatite binding. 17 Due to the high collagen concentration in the composite solution, the collagen does not perfectly combine with the hydroxyapatite. Greater the collagen concentration in a solution, the smaller the diameter produced (Table 2). Because collagen is a polyelectrolyte or an ionized linear polymer with a large number of functional groups, its presence can increase the conductivity of polymer solutions. The resulting ber will be more delicate and have a smaller diameter as the conductivity of the solution increases. When the ber diameter decreases, the bers are oriented more randomly, resulting in a lower ber density and a larger empty area (Table 3).

Mechanical properties of PCL-HA-collagen ber scaffold
Mechanical properties were determined on both braided and non-braided samples. Table 4 shows the values of Ultimate Tensile Strength (UTS), modulus of elasticity, and elongation in the samples. The lower the UTS value and the higher the elastic modulus, the lower the HA composition and the higher the collagen composition. The ultimate tensile strength of ACL is  approximately 36 MPa. 18 Furthermore, the modulus of elasticity of ACL ranged from 65 to 111 ± 29 MPa. 19 The UTS value and modulus of elasticity do not meet the human ACL standard, as shown in Fig. 6 and 7. This could be due to the random orientation of the bers. Randomly oriented bers cannot withstand stress in the same direction, resulting in low mechanical strength. 20 In the braiding sample, the UTS value and modulus of elasticity were lower. This could be due to the braiding process being done by hand, resulting in less tight braids; as a result, the mechanical strength was lower than that of the non-braiding one (Fig. 5). 21

PCL-HA-collagen scaffold degradation rate
One of the most important factors in tissue engineering is the rate of degradation. To determine whether the scaffold is still available or has been completely degraded during the tissue growth process, the degradation rate must be measured. Fig. 7 shows the results of the mass of the degraded samples (in %) aer 7, 14, 21, and 28 days. According to the linear regression results in Fig. 7, the rate of degradation of each sample corresponds to the gradient value of each regression equation. Based on the regression equation, it is also possible to predict when the entire scaffold will be completely degraded, assuming a constant degradation rate. Table 5 displays the results of the calculation of the degradation rate and the estimated time-out for each sample. The degradation rate of the scaffold must correspond to the formation of ligaments. The rate of cell proliferation will be disrupted if degradation occurs too rapidly. In contrast, if the rate of degradation is too slow, it will interfere with the tissue's biological function. 22

PCL-HA-collagen scaffold cell viability
As part of tissue engineering, a scaffold must be non-toxic. Using Baby Hamster Kidney (BHK-21) cells, a cytotoxicity test was conducted using the MTT assay method. The MTT assay is based    on the principle that cells with metabolic activity reduce MTT salts through the work of enzymes. When the enzyme reacts with MTT, a purple formazan will be produced. The absorbance of living cells will be determined by spectrometry measurements of the intensity of this purple hue. Fig. 8 illustrates the results of the live cell count. Sample E with a PCL-HA-collagen ratio of 50 : 25 : 25 exhibited the highest cell viability. This result is the result of multiple factors; in the sample, the addition of collagen, which can facilitate broblast growth and tissue regeneration, allows BHK cells to perform cell activity more effectively.

Discussion
The FTIR spectrum revealed the presence of PCL, hydroxyapatite, and collagen marker groups based on functional group analysis. Because the FTIR spectrum results do not offer any new functional groups apart from the functional groups of the three materials, these three materials are only physically mixed. The functional groups are the HA PO 4 3− a group, the collagen amide III (N-H bend), and the carbonyl group of the C]O stretch group. As a result, the synthesis of PCL-HA-collagen scaffold in the form of ber via the electrospinning process was deemed successful. 16 PCL samples containing hydroxyapatite and collagen had a rougher surface morphology than PCL samples lacking hydroxyapatite and collagen. Further analysis using ImageJ soware revealed that the average ber diameter ranged between 365 and 488 nm ( Table 2). Nanostructures have a high surface area ratio, allowing for more cell attachment space than other structures. Furthermore, bers with a diameter of 1000 nm can increase the activity of cells in forming an extracellular matrix. 23  Mechanical strength is an important property of scaffolding, especially for ACL. The ACL is a ligament that acts as a back and front movement barrier as well as a knee stabilizer. To perform this function, you must have a high modulus of elasticity and a low UTS. Similarly, one of the parameters that must be considered on the ACL scaffold is the mechanical property. Mechanical strength (modulus of elasticity and UTS) increased with increasing hydroxyapatite composition in PCL : HA : collagen scaffold samples. However, the results are still insuf-cient to match the mechanical strength of the human ACL. The addition of hydroxyapatite to collagen can increase the scaffold's modulus of elasticity and UTS. 24 Braiding approach should be able to improve the mechanical properties of the scaffold. 13,25 However, our braiding process has failed to increase the mechanical strength value. This is presumably due to the fact that the process is done manually. Therefore, one bond is not perfectly interwoven with another, in contrast with previous report of braided scaffold fabricated using braiding machine to form a perfect braided scaffold. 26 The presence of collagen in the sample causes the degradation rate to be faster, resulting in a higher quality of mass degraded in the sample. This is due to the fact that collagen interacts with water more easily than PCL and HA. Furthermore, collagen is a polymer with amorphous properties. Ligaments can regenerate for a period of 6-8 months. As a result, in this study, the appropriate degraded samples were C, D, and E. 27 A live cell percentage of more than 60% is required for tissue engineering. 28 The samples in this study had a percentage value of living cells above 60%, indicating that the sample does not have toxic properties. Based on the results of the above characterizations, the PCL : HA : collagen ber scaffold has the potential as an ACL scaffold. However, mechanical strength needs to be increased.

Conclusion
Variations in HA and collagen composition inuence ber diameter and morphology, porosity, and mass loss percentage. The samples with a high hydroxyapatite concentration still contained beads. As the concentration of collagen increases, no beads form. As the average diameter of all samples is less than 1000 nm, cell attachment is facilitated. The percentage of mass lost increases as collagen levels rise. The sample is non-toxic in the cytotoxicity test because the percentage of viable cells is greater than 60%. The optimal composition is found in samples with a ratio of 50 : 45 : 5 PCL : HA : collagen. This sample's ber diameter ranges from 203 to 1535 nm, with a mean ber diameter of 488 ± 271 nm. The UTS value and modulus of elasticity for the braided sample were 2.796 MPa and 3.224 MPa, whereas they were 2.864 MPa and 12.942 MPa, respectively, for the unbraided sample. They estimated a total of 9.44 months of mass exhaustion. It contains 87.95% of living cells and is nontoxic.

Conflicts of interest
The authors declare no conict of interest.